Hypoxia, the state of reduced oxygen levels, triggers a multifaceted adaptive response mediated by oxygen- dependent transcription factors, termed hypoxia-inducible factors (HIFs). Among these, hypoxia-Inducible Factor 1 (HIF1) is the main regulator of oxygen-dependent transcription in a majority of organs and accounts for the increase in expression of hypoxia-inducible genes. HIF controls an expression of over 100 genes involved in angiogenesis, altered energy metabolism, anti-apoptotic, and pro-proliferative mechanisms. Elevated levels of HIF1 are an independent, prognostic factor for a diverse range of malignant neoplasms. We are developing small molecules chemical tools that provide exquisite control over transcriptional activation of hypoxia-inducible genes implicated in cancer progression. Specifically, we will target three key signaling events mediated by three distinct protein-protein interactions with unique synthetic ligands. Small molecules often interact non-specifically with large protein surfaces; and hence inhibition of protein-protein interactions with small molecules remains difficult. Nevertheless, we recently demonstrated that hypoxia-inducible signaling can be effectively modulated in vitro and in vivo by rationally designed dimeric epidithiodiketopiperazines (ETPs) - ligands that bind p300/CBP CH1 region, induce structural change to these domains and prevent it from interacting with HIF1? C-terminal transactivation domain. The specific hypothesis behind the proposed research is that tumor progression could be effectively down-regulated by targeting of HIF1? C-TAD in complex with the CH1 domain of the coactivator protein p300/CBP with these designed molecules. Specific Aim 1 focuses on synthesis of conformational mimetics of the dimeric epidithiodiketopiperazine (ETP) natural products, characterization of the ETPs binding properties via isothermal titration microcalorimetry (ITC), surface plasmon resonance (SPR), and fluorescence polarization competition experiments. In Specific Aim 2 we will evaluate the stability of dimeric ETPs in cell culture and test efficacy in inhibiting HIF-inducible transcription with luciferase assays, qRT-PCR, ELISA. In this Aim we will also investigate structural basis of the inhibition of HIF1?-coactivator interactions by employing site- directed mutagenesis and structural NMR. In Specific Aim 3 we will explore mechanistic framework of ETPs with pathway-based RT2 Profiler assays, gene expression profiling, and pathway analysis. We will also conduct in vivo experiments in order to determine efficacy of the ETPs in our mouse tumor xenograft models. Combined the three aims will validate our hypothesis and could lead to novel, unique tools for dissecting the hypoxia-inducible signaling pathway in cancer and facilitate development of future therapeutics.